Human GTP-binding proteins

ABSTRACT

The present invention provides three novel GTP-binding proteins (designated individually as BND-1, BND-2, and BND-3, and collectively as BND) and polynucleotides which identify and encode BND. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding BND and a method for producing BND. The invention also provides for use of BND and agonists, antibodies, or antagonists specifically binding BND, in the prevention and treatment of diseases associated with expression of BND. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding BND for the treatment of diseases associated with the expression of BND. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding BND.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of threenovel GTP-binding proteins and to the use of these sequences in thediagnosis, prevention, and treatment of disorders and diseases ofabnormal cellular proliferation and signal transduction.

BACKGROUND OF THE INVENTION

Guanine nucleotide binding proteins (GTP-binding proteins) participatein a wide range of regulatory functions in all organisms. They arepresent in all eukaryotic cells and function in processes includingmetabolism, cellular growth, differentiation, signal transduction,cytoskeletal organization, and intracellular vesicle transport andsecretion. The GTP-binding proteins control a diverse sets of regulatorypathways and consequently, play a key role in the cell's ability toprocess and respond to information. Much of this information is providedto individual cells in the form of changes in concentration of hormones,growth factors, neuromodulators, or other molecules. When thesemolecules bind to transmembrane receptors, a signal is propagated toeffector molecules by intracellular signal transducing proteins, many ofwhich are members of the G-protein family.

The superfamily of GTP-binding proteins consists of several familiesincluding translational factors, heterotrimeric GTP-binding proteinsinvolved in transmembrane signaling processes, protooncogenic rasproteins, and low-molecular weight (ras family) GTP-binding proteins.

Heterotrimeric GTP-binding proteins are composed of 3 subunits (α, β andγ) which, in the resting state, associate as a trimer at the inner faceof the plasma membrane. The α subunits has a molecule of guanosinediphosphate (GDP) bound to it: stimulation of the G-protein by anactivated receptor leads to its exchange for guanosine triphosphate(GTP). This results in the separation of the α from the β and γsubunits, which remain tightly associated as a dimer. Both the α and β-γsubunits are then able to interact with effectors, either individuallyor in a cooperative manner. The intrinsic GTPase activity of the αsubunits hydrolyses the bound GTP to GDP. This returns the α subunit toits inactive conformation and allows it to reassociate with the β-γsubunits, which restores the system to its resting state (Kaziro, Y.(1991) Annu. Rev. Biochem. 60:349-400;).

Many distinct classes of α, β, and γ subunits have been identified inmammalian heterotrimeric GTP-binding proteins. The α-s class issensitive to ADP-ribosylation by pertussis toxin which uncouples thereceptor:G protein interaction. This uncoupling blocks signaltransduction to receptors that decrease cAMP levels, which regulates ionchannels and activates phospholipases. The inhibitory, α-I class, isalso susceptible to modification by pertussis toxin which prevents α-Ifrom lowering cAMP levels. Two novel classes refractory to pertussistoxin modification, are α-q which activates phospholipase C and α-12which has sequence homology with the Drosophila gene concertina and maycontribute to the regulation of embryonic development (Simon, M. I.(1991) Science 252:802-808). The β subunit sequences are highlyconserved between species implying that they perform a fundamentallyimportant role in the organization and function of G-protein linkedsystems (Van der Voorn L. (1992) Febs. Lett. 307 (2):131-134). The γsubunit primary structures are more variable than those of the γsubunits. They are often post-translationally modified by isoprenylationand carboxyl-methylation of a cysteine residue 4 amino acids from theC-terminus; this appears to be necessary for the interaction of the β-γsubunit with the membrane and with other GTP-binding proteins. The β-γsubunit has been shown to modulate the activity of isoforms of adenylylcyclase, phospholipase C, and some ion channels. It is involved inreceptor phosphorylation via specific kinases, and has been implicatedin the p21ras-dependent activation of the MAP kinase cascade and therecognition of specific receptors by the GTP-binding proteins. (Clapham,D. E. (1993) Nature 365:403-406).

The low molecular weight GTP-binding proteins regulate cell growth, cellcycle control, protein secretion, and intracellular vesicle interaction.At least sixty members of this ras-related superfamily have beenidentified and are currently grouped into the four subfamilies of ras,rho, ran, and rab. They consist of single polypeptides of 21-30 kDwhich, like the α subunit of the heterotrimeric GTP-binding proteins,are able to bind and to hydrolyze GTP, thus cycling from an inactive toan active state. These GTP-binding proteins respond to extracellularsignals from receptors and activating proteins by transducing mitogenicsignals involved in various cell functions. (Tavitian, A. (1995) C. R.Seances Soc. Biol. Fil. 189:7-12).

Activated ras genes were initially found in human cancers and subsequentstudies confirmed that ras function is critical in the determination ofwhether cells continue to grow or become terminally differentiated.Stimulation of cell surface receptors activates ras which, in turn,activates cytoplasmic kinases. The kinases translocate to the nucleusand activate key transcription factors that control gene expression andprotein synthesis. (Barbacid, M. (1987)Ann. Rev Biochem. 56:779-827,Treisman, R. (1994) Curr. Opin. Genet. Dev. 4:96-98).

The other members of the small G-protein superfamily have roles insignal transduction that vary with the function of the activated genesand the locations of the GTP-binding proteins that initiate theactivity. The rho GTP-binding proteins control signal transductionpathways that link growth factor receptors to actin polymerization whichis necessary for normal cellular growth and division. The rab proteinscontrol the translocation of vesicles to and from membranes for proteinlocalization, protein processing, and secretion. The ran GTP-bindingproteins are located in the nucleus of cells and have a key role innuclear protein import, the control of DNA synthesis, and cell-cycleprogression (Hall, A. (1990) Science 249:635-640, Scheffzek, K. et al.(1995) Nature 374:378-381).

The discovery of polynucleotides encoding new GTP-binding proteins, andthe molecules themselves, provides the means to investigate abnormalcell proliferation and signal transduction processes. Discovery of novelmolecules related to human GTP-binding proteins and the polynucleotidesencoding them satisfies a need in the art by providing new diagnostic ortherapeutic compositions useful in diagnosis, prevention, and treatmentof disorders and diseases of abnormal cellular proliferation and signaltransduction.

SUMMARY OF THE INVENTION

The present invention features three novel human GTP-binding proteins,designated individually as BND-1, BND-2 and BND-3 and collectively asBND, and characterized as having similarity to human G25K, human rab30,and mouse GTP-binding protein.

Accordingly, the invention features substantially purified BND-1, BND-2,and BND-3 having the anino acid sequences; SEQ ID NO:1, SEQ ID NO:3, andSEQ ID NO:5, respectively.

One aspect of the invention features isolated and substantially purifiedpolynucleotides that encode BND-1, BND-2, and BND-3. In a particularaspect, the polynucleotides are the nucleotide sequences of SEQ ID NO:2,SEQ ID NO:4, or SEQ ID NO:6, respectively.

The invention also features a polynucleotide sequence comprising thecomplement of SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6, or variantsthereof. In addition, the invention features polynucleotide sequenceswhich hybridize under stringent conditions to SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6.

The invention additionally features nucleic acid sequences encodingpolypeptides, oligonucleotides, peptide nucleic acids (PNA), fragments,portions or antisense molecules thereof, and expression vectors and hostcells comprising polynucleotides that encode BND. The present inventionalso features antibodies which bind specifically to BND, andpharmaceutical compositions comprising substantially purified BND. Theinvention also features the use of agonists and antagonists of BND.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A and 1B show the amino acid sequence (SEQ ID NO:1) and nucleicacid sequence (SEQ ID NO:2) of BND-1. The alignment was produced usingMACDNASIS PRO™ software (Hitachi Software Engineering Co., Ltd., SanBruno, Calif.).

FIGS. 2A and 2B show the amino acid sequence (SEQ ID NO:3) and nucleicacid sequence (SEQ ID NO:4) of BND-2.

FIGS. 3A and 3B show the amino acid sequence (SEQ ID NO:5) and nucleicacid sequence (SEQ ID NO:6) of BND-3.

FIG. 4 shows the amino acid sequence alignment between BND-1 (SEQ IDNO:1) and human G25K (GI 183490). The alignment was produced using themultisequence alignment program of DNASTAR™ software (DNASTAR Inc,Madison, Wis.).

FIG. 5 shows the amino acid sequence alignment between BND-2 (SEQ IDNO:3) and human rab30 (GI 1457955).

FIG. 6 shows the amino acid sequence alignment between BND-3 (SEQ IDNO:5) and mouse GTP-binding protein (GI 240986).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a", "an", and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, reference to "ahost cell" includes a plurality of such host cells, reference to the"antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

DEFINITIONS

Nucleic acid sequence, as used herein, refers to an oligonucleotide,nucleotide, or polynucleotide, and fragments or portions thereof, and toDNA or RNA of genomic or synthetic origin which may be single- ordouble-stranded and represent the sense or antisense strand. Similarly,"amino acid sequence", as used herein, refers to an oligopeptide,peptide, polypeptide, or protein sequence, and fragments or portionsthereof, and to naturally occurring or synthetic molecules.

Where "amino acid sequence" is recited herein to refer to an amino acidsequence of a naturally occurring protein molecule, "amino acidsequence" and like terms, such as "polypeptide" or "proteins" are notmeant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule. "Peptidenucleic acid", as used herein, refers to a molecule which comprises anoligomer to which an amino acid residue, such as lysine, and an aminogroup have been added. These small molecules, also designated anti-geneagents, stop transcript elongation by binding to their complementarystrand of nucleic acid (Nielsen, P. E. et al. (1993) Anticancer DrugDes. 8:53-63).

BND, as used herein, refers to the amino acid sequences of substantiallypurified BND obtained from any species, particularly mammalian,including bovine, ovine, porcine, murine, equine, and preferably human,from any source whether natural, synthetic, semi-synthetic, orrecombinant.

"Consensus", as used herein, refers to a nucleic acid sequence which hasbeen resequenced to resolve uncalled bases, or which has been extendedusing XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

A "variant" of BND, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have"conservative" changes, wherein a substituted amino acid has similarstructural or chemical properties, e.g., replacement of leucine withisoleucine. More rarely, a variant may have "nonconservative" changes,e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

A "deletion", as used herein, refers to a change in either amino acid ornucleotide sequence in which one or more amino acid or nucleotideresidues, respectively, are absent.

An "insertion" or "addition", as used herein, refers to a change in anamino acid or nucleotide sequence resulting in the addition of one ormore amino acid or nucleotide residues, respectively, as compared to thenaturally occurring molecule.

A "substitution", as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

The term "biologically active", as used herein, refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic BND, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The term "agonist", as used herein, refers to a molecule which, whenbound to BND, causes a change in BND which modulates the activity ofBND. Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to BND.

The terms "antagonist" or "inhibitor", as used herein, refer to amolecule which, when bound to BND, blocks or modulates the biological orimmunological activity of BND. Antagonists and inhibitors may includeproteins, nucleic acids, carbohydrates, or any other molecules whichbind to BND.

The term "modulate", as used herein, refers to a change or an alterationin the biological activity of BND. Modulation may be an increase or adecrease in protein activity, a change in binding characteristics, orany other change in the biological, functional or immunologicalproperties of BND.

The term "mimetic", as used herein, refers to a molecule, the structureof which is developed from knowledge of the structure of BND or portionsthereof and, as such, is able to effect somc or all of the actions ofG-protein-like molecules.

The term "derivative", as used herein, refers to the chemicalmodification of a nucleic acid encoding BND or the encoded BND.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

The term "substantially purified", as used herein, refers to nucleic oramino acid sequences that are removed from their natural environment,isolated or separated, and are at least 60% free, preferably 75% free,and most preferably 90% free from other components with which they arenaturally associated.

"Amplification" as used herein refers to the production of additionalcopies of a nucleic acid sequence and is generally carried out usingpolymerase chain reaction (PCR) technologies well known in the art(Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primers, a LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y.).

The term "hybridization", as used herein, refers to any process by whicha strand of nucleic acid binds with a complementary strand through basepairing.

The term "hybridization complex", as used herein, refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen binds between complementary G and C bases and betweencomplementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀ t or R₀ tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

The terms "complementary" or "complementarity", as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base-pairing. For example, for the sequence"A-G-T" binds to the complementary sequence "T-C-A". Complementaritybetween two single-stranded molecules may be "partial", in which onlysome of the nucleic acids bind, or it may be complete when totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of hybridization between nucleic acidstrands. This is of particular importance in amplification reactions,which depend upon binding between nucleic acids strands.

The term "similarity", as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete similarity(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term"substantially similar." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially similar sequence or probe will compete for and inhibit thebinding (i.e., the hybridization) of a completely homologous sequence orprobe to the target sequence under conditions of low stringency. This isnot to say that conditions of low stringency are such that non-specificbinding is permitted; low stringency conditions require that the bindingof two sequences to one another be a specific (i.e., selective)interaction. The absence of non-specific binding may be tested by theuse of a second target sequence which lacks even a partial degree ofcomplementarity (e.g., less than about 30% identity); in the absence ofnon-specific binding, the probe will not hybridize to the secondnoncomplementary target sequence.

As known in the art, numerous equivalent conditions may be employed tocomprise either low or high stringency conditions. Factors such as thelength and nature (DNA, RNA, base composition) of the sequence, natureof the target (DNA, RNA, base composition, presence in solution orimmobilization, etc.), and the concentration of the salts and othercomponents (e.g., the presence or absence of formamide, dextran sulfateand/or polyethylene glycol) are considered and the hybridizationsolution may be varied to generate conditions of either low or highstringency different from, but equivalent to, the above listedconditions.

The term "stringent conditions", as used herein, is the "stringency"which occurs within a range from about Tm-5° C. (5° C. below the meltingtemperature (Tm) of the probe) to about 20° C. to 25° C. below Tm. Aswill be understood by those of skill in the art, the stringency ofhybridization may be altered in order to identify or detect identical orrelated polynucleotide sequences.

The term "antisense", as used herein, refers to nucleotide sequenceswhich are complementary to a specific DNA or RNA sequence. The term"antisense strand" is used in reference to a nucleic acid strand that iscomplementary to the "sense" strand. Antisense molecules may be producedby any method, including synthesis by ligating the gene(s) of interestin a reverse orientation to a viral promoter which permits the synthesisof a complementary strand. Once introduced into a cell, this transcribedstrand combines with natural sequences produced by the cell to formduplexes. These duplexes then block either the further transcription ortranslation. In this manner, mutant phenotypes may be generated. Thedesignation "negative" is sometimes used in reference to the antisensestrand, and "positive" is sometimes used in reference to the sensestrand.

The term "portion", as used herein, with regard to a protein (as in "aportion of a given protein") refers to fragments of that protein. Thefragments may range in size from four amino acid residues to the entireamino acid sequence minus one amino acid. Thus, a protein "comprising atleast a portion of the amino acid sequence of SEQ ID NO:1" encompassesthe full-length human BND-1 and fragments thereof.

"Transformation", as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such"transformed" cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

The term "antigenic determinant", as used herein, refers to that portionof a molecule that makes contact with a particular antibody (i.e., anepitope). When a protein or fragment of a protein is used to immunize ahost animal, numerous regions of the protein may induce the productionof antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

The terms "specific binding" or "specifically binding", as used herein,in reference to the interaction of an antibody and a protein or peptide,mean that the interaction is dependent upon the presence of a particularstructure (i.e., the antigenic determinant or epitope) on the protein;in other words, the antibody is recognizing and binding to a specificprotein structure rather than to protein in general. For example, if anantibody is specific for epitope "A", the presence of a proteincontaining epitope A (or free, unlabeled A) in a reaction containinglabeled "A" and the antibody will reduce the amount of labeled A boundto the antibody.

The term "sample", as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acid encoding BND orfragments thereof may comprise a cell, chromosomes isolated from a cell(e.g., a spread of metaphase chromosomes), genomic DNA (in solution orbound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

The term "correlates with expression of a polynucleotide", as usedherein, indicates that the detection of the presence of ribonucleic acidthat is similar to SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 by northernanalysis is indicative of the presence of mRNA encoding BND in a sampleand thereby correlates with expression of the transcript from thepolynucleotides encoding the protein.

"Alterations" in the polynucleotide of SEQ ID NO:2, SEQ ID NO:4, or SEQID NO:6, as used herein, comprise any alteration in the sequence ofpolynucleotides encoding BND including deletions, insertions, and pointmutations that may be detected using hybridization assays. Includedwithin this definition is the detection of alterations to the genomicDNA sequence which encodes BND (e.g., by alterations in the pattern ofrestriction fragment length polymorphisms capable of hybridizing to SEQID NO:2, SEQ ID NO:4, or SEQ ID NO:6), the inability of a selectedfragment of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding BND (e.g., using fluorescent in situhybridization (FISH) to metaphase chromosomes spreads).

As used herein, the term "antibody" refers to intact molecules as wellas fragments thereof, such as Fa, F(ab)₂, and Fv, which are capable ofbinding the epitopic determinant. Antibodies that bind BND polypeptidescan be prepared using intact polypeptides or fragments containing smallpeptides of interest as the immunizing antigen. The polypeptide orpeptide used to immunize an animal can be derived from the translationof mRNA or synthesized chemically, and can be conjugated to a carrierprotein, if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin and thyroglobulin. The coupledpeptide is then used to immunize the animal (e.g., a mouse, a rat, or arabbit).

The term "humanized antibody", as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

THE INVENTION

The invention is based on the discovery of novel human GTP-bindingproteins (BND-1, BND-2, and BND-3, collectively referred to as BND), thepolynucleotides encoding BND, and the use of these compositions for thediagnosis, prevention and treatment of disorders and diseases ofabnormal cellular proliferation and signal transduction.

Nucleic acid sequence encoding the human BND-1 of the present inventionwas first identified in Incyte Clone 113700 from a testicular tissuecDNA library (TESTNOT01) through a computer-generated search for aminoacid sequence alignments. A consensus sequence, SEQ ID NO:2, was derivedfrom the following overlapping and/or extended nucleic acid sequences(cDNA library from which derived): Incyte Clones 113700 (TESTNOT01) and179322 (PLACNOB01).

Nucleic acid sequence encoding the human BND-2 of the present inventionwas first identified in Incyte Clone 583177 from a prostate tissue cDNAlibrary (PROSNOT02) through a computer-generated search for amino acidsequence alignments. A consensus sequence, SEQ ID NO:4, was derived fromthe following overlapping and/or extended nucleic acid sequences (andcDNA library from which derived): Incyte Clones 583177 and 582201(PROSNOT02); 899921 and 902624 (BRSTTUT03); 992064 (COLNNOT 11); 078316(SYNORAB01); and 1424482 (BEPINON01).

Nucleic acid sequence encoding the human BND-3 of the present inventionwas first identified in Incyte Clone 627051, from a paraganglionic tumortissue cDNA library (PGANNOT01) through a computer-generated search foramino acid sequence alignments. A consensus sequence, SEQ ID NO:6, wasderived from the following overlapping and/or extended nucleic acidsequences (cDNA library from which derived): Incyte Clones 627051(PGANNOT01); 586525 (UTRSNOT01); 719363 (SYNOOAT01); 836241 and 837631(PROSNOT07); 1228396 (COLNOT01); 1316240 (BLADTUT02); 1378809(LUNGNOT01); 855922 and 856810 (NGANNOT01); 929744 (CERVNOT01); 628180and 955099 (KIDNNOT05); 994996 and 1001874 (BRSTNOT03); and 147085(FIBRNOT02).

In one embodiment, the invention encompasses the novel human GTP-bindingprotein BND-1, a polypeptide comprising the amino acid sequence of SEQID NO:1, and shown in FIGS. 1A, 1B. BND-1 is 182 amino acids in lengthand, as shown in FIG. 4, BND-1 has chemical and structural homology withhuman G25K (GI 183490). In particular, BND-1 shares 53% identity with G183490. BND-1 contains two GTP binding domains spanning amino acids L₃₈to Q₄₆ and L₉₇ to L₁₀₄. The amino acid motif which defines thesedomains, consisting of a P-loop structure, is conserved between BIND-1and GI 183490.

In another embodiment, the invention encompasses the novel humanGTP-binding protein BND-2, a polypeptide comprising the amino acidsequence of SEQ ID NO:3, as shown in FIGS. 2A, 2B. BND-2 is 186 aminoacids in length and, as shown in FIG. 5, BND-2 has chemical andstructural homology with human rab30 (GI 1020151). In particular, BND-2shares 32% homology with GI 1020151. BND-2 contains three GTP bindingdomains spanning amino acids V₁₆ to T₁₉, V₁₁₉, to D₁₂₆, and E₁₅₁ toK₁₅₅, which are also seen in GI 1020151.

In an additional embodiment, the invention encompasses the novel humanGTP-binding proteins BND-3, a polypeptide comprising the amino acidsequence of SEQ ID NO:5, as shown in FIGS. 3A, 3B. BND-3 is 169 aminoacids in length and, as shown in FIG. 6, BND-3 has chemical andstructural homology with mouse GTP-binding protein (GI 240986). Inparticular, BND-3 and GI 240986 share 74% identity. BND-3 contains twoGTP binding domains defined by amino acids L₁₃ to F₂₄ and F₇₂ to L₈₀which are also seen in GI 240986.

The invention also encompasses BND variants. A preferred BND variant isone having at least 80%, and more preferably 90%, amino acid sequencesimilarity to the BND amino acid sequence (SEQ ID NO:1, SEQ ID NO:3, orSEQ ID NO:5). A most preferred BND variant is one having at least 95%amino acid sequence similarity to SEQ ID NO:1, SEQ ID NO:3, or SEQ IDNO:5.

The invention also encompasses polynucleotides which encode BND.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of BND can be used to generate recombinant molecules whichexpress BND. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid of SEQ ID NO:2, SEQ ID NO:4,or SEQ ID NO:6 as shown in FIGS. 1A and 1B, 2A and 2B, and 3A and 3B.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of nucleotide sequencesencoding BND, some bearing minimal homology to the nucleotide sequencesof any known and naturally occurring gene, may be produced. Thus, theinvention contemplates each and every possible variation of nucleotidesequence that could be made by selecting combinations based on possiblecodon choices. These combinations are made in accordance with thestandard triplet genetic code as applied to the nucleotide sequence ofnaturally occurring BND, and all such variations are to be considered asbeing specifically disclosed.

Although nucleotide sequences which encode BND and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring BND under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding BND or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding BND and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences, or portionsthereof, which encode BND and its derivatives, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art at the time of the filing ofthis application. Moreover, synthetic chemistry may be used to introducemutations into a sequence encoding BND or any portion thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed nucleotide sequences, and inparticular, those shown in SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,under various conditions of stringency. Hybridization conditions arebased on the melting temperature (Tm) of the nucleic acid bindingcomplex or probe, as taught in Wahl, G. M. and S. L. Berger (1987;Methods Enzymol. 152:399-407) and Kimmel, A. R. (1987; Methods Enzymol.152:507-11), and may be used at a defined stringency.

Altered nucleic acid sequences encoding BND which are encompassed by theinvention include deletions, insertions, or substitutions of differentnucleotides resulting in a polynucleotide that encodes the same or afunctionally equivalent BND. The encoded protein may also containdeletions, insertions, or substitutions of amino acid residues whichproduce a silent change and result in a functionally equivalent BND.Deliberate amino acid substitutions may be made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues as long asthe biological activity of BND is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid;positively charged amino acids may include lysine and arginine; andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine;phenylalanine and tyrosine.

Also included within the scope of the present invention are alleles ofthe gene encoding BND. As used herein, an "allele" or "allelic sequence"is an alternative form of the gene which may result from at least onemutation in the nucleic acid sequence. Alleles may result in alteredmRNAs or polypeptides whose structure or function may or may not bealtered. Any given gene may have none, one, or many allelic forms.Common mutational changes which give rise to alleles are generallyascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

Methods for DNA sequencing which are well known and generally availablein the art may be used to practice any embodiments of the invention. Themethods may employ such enzymes as the Kienow fragment of DNA polymeraseI, Sequenase® (US Biochemical Corp, Cleveland, Ohio), Taq polymerase(Perkin Elmer), thermostable T7 polymerase (Amersham, Chicago, Ill.), orcombinations of recombinant polymerases and proofreading exonucleasessuch as the ELONGASE Amplification System marketed by Gibco BRL(Gaithersburg, Md.). Preferably, the process is automated with machinessuch as the Hamilton Micro Lab 2200 (Hamilton, Reno, Nev.), PeltierThermal Cycler (PTC200; MJ Research, Watertown, Mass.) and the ABI 377DNA sequencers (Perkin Elmer).

The nucleic acid sequences encoding BND may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences such as promoters and regulatoryelements. For example, one method which may be employed,"restriction-site" PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO®4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

Another method which may be used to retrieve unknown sequences is thatof Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDER™libraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will contain moresequences which contain the 5' regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5' and 3'non-transcribed regulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and detection of the emitted wavelengths by a charge coupled devicecamera. Output/light intensity may be converted to electrical signalusing appropriate software (e.g. GENOTYPER™ and SEQUENCE NAVIGATOR™,Perkin Elmer) and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for the sequencing ofsmall pieces of DNA which might be present in limited amounts in aparticular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode BND, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of BND in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressBND.

As will be understood by those of skill in the art, it may beadvantageous to produce BND-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter sequencesencoding BND for a variety of reasons, including but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, to alterglycosylation patterns, to change codon preference, to produce splicevariants, or to introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant polynucleotides encoding BND may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of BND activity, it may be useful toencode a chimeric BND protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between a sequence encoding BND and theheterologous protein sequence, so that BND may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding BND may be synthesized, inwhole or in part, using chemical methods well known in the art (seeCaruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser. 215-223,Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of BND, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, WH Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of BND, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

In order to express a biologically active BND, the nucleotide sequencesencoding BND or functional equivalents, may be inserted into appropriateexpression vectors, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding BND andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

A variety of expression vector/host systems may be utilized to containand express sequences encoding BND. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

The "control elements" or "regulatory sequences" are thosenon-translated regions of the vector--enhancers, promoters, 5' and 3'untranslated regions--which interact with host cellular proteins tocarry out transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT® phagemid (Stratagene,LaJolla, Calif.) or PSPORTI™ plasmid (Gibco BRL), and the like, may beused. The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding BND,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for BND. For example, when largequantities of BND are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to, themultifunctional E. coli cloning and expression vectors such asBLUESCRIPT® (Stratagene), in which the sequence encoding BND may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast, Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters such as alpha factor, alcoholoxidase, and PGH may be used. For reviews, see Ausubel et al. (supra)and Grant et al. (1987) Methods Enzymol. 153:516-544.

In cases where plant expression vectors are used, the expression of asequence encoding BND may be driven by any of a number of promoters. Forexample, viral promoters such as the 35S and 19S promoters of CaMV maybe used alone or in combination with the omega leader sequence from TMV(Takamatsu, N. (1987) EMBO J. 6:307-311). Alternatively, plant promoterssuch as the small subunits of RUBISCO or heat shock promoters may beused (Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680; Broglie, R. et al.(1984) Science 224:838-843; and Winter, J. et al. (1991) Results Probl.Cell Differ. 17:85-105). These constructs can be introduced into plantcells by direct DNA transformation or pathogen-mediated transfection.Such techniques are described in a number of generally available reviews(see, for example, Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook ofScience and Technology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

An insect system may also be used to express BND. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding BND may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of BND will render the polyhedrin gene inactive and producerecombinant virus lacking coat protein. The recombinant viruses may thenbe used to infect, for example, S. frugiperda cells or Trichoplusialarvae in which BND may be expressed (Engelhard, E. K. et al. (1994)Proc. Nat. Acad. Sci. 91:3224-3227).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding BND may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing BND in infected host cells (Logan, J. and Shenk,T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding BND. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding BND, its initiation codon, and upstream sequences are insertedinto the appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a portion thereof, is inserted, exogenoustranslational control signals including the ATG initiation codon shouldbe provided. Furthermore, the initiation codon should be in the correctreading frame to ensure translation of the entire insert. Exogenoustranslational elements and initiation codons may be of various origins,both natural and synthetic. The efficiency of expression may be enhancedby the inclusion of enhancers which are appropriate for the particularcell system which is used, such as those described in the literature(Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162).

In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andW138, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressBND may be transformed using expression vectors which may contain viralorigins of replication and/or endogenous expression elements and aselectable marker gene on the same or on a separate vector. Followingthe introduction of the vector, cells may be allowed to grow for 1-2days in an enriched media before they are switched to selective media.The purpose of the selectable marker is to confer resistance toselection, and its presence allows growth and recovery of cells whichsuccessfully express the introduced sequences. Resistant clones ofstably transformed cells may be proliferated using tissue culturetechniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, its presence and expression mayneed to be confirmed. For example, if the sequence encoding BND isinserted within a marker gene sequence, recombinant cells containingsequences encoding BND can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding BND under the control of a single promoter. Expressionof the marker gene in response to induction or selection usuallyindicates expression of the tandem gene as well.

Alternatively, host cells which contain sequences encoding andexpressing BND may be identified by a variety of procedures known tothose of skill in the art. These procedures include, but are not limitedto, DNA--DNA or DNA-RNA hybridizations and protein bioassay orimmunoassay techniques which include membrane, solution, or chip basedtechnologies for the detection and/or quantification of the nucleic acidor protein.

The presence of polynucleotide sequences encoding BND can be detected byDNA--DNA or DNA-RNA hybridization or amplification using probes orportions or fragments of polynucleotides encoding BND. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding BND to detect transformantscontaining DNA or RNA encoding BND. As used herein "oligonucleotides" or"oligomers" refer to a nucleic acid sequence of at least about 10nucleotides and as many as about 60 nucleotides, preferably about 15 to30 nucleotides, and more preferably about 20-25 nucleotides, which canbe used as a probe or amplimer.

A variety of protocols for detecting and measuring the expression ofBND, using either polyclonal or monoclonal antibodies specific for theprotein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson BND is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding BND includeoligolabeling, nick translation, end-labeling or PCR amplification usinga labeled nucleotide. Alternatively, sequences encoding BND, or anyportion thereof, may be cloned into a vector for the production of anmRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits from Pharmacia & Upjohn (Kalamazoo, Mich.);Promega (Madison, Wis.); and U.S. Biochemical Corp. (Cleveland, Ohio).Suitable reporter molecules or labels, which may be used, includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents as well as substrates, cofactors, inhibitors, magnetic particles,and the like.

Host cells transformed with nucleotide sequences encoding BND may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a recombinantcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeBND may be designed to contain signal sequences which direct secretionof BND through a prokaryotic or eukaryotic cell membrane. Otherrecombinant constructions may be used to join sequences encoding BND tonucleotide sequence encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and BND may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingBND and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMIAC (immobilized metal ion affinitychromatography) as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3: 263-281) while the enterokinase cleavage site provides a meansfor purifying BND from the fusion protein. A discussion of vectors whichcontain fusion proteins is provided in Kroll, D. J. et al. (1993; DNACell Biol. 12:441-453).

In addition to recombinant production, fragments of BND may be producedby direct peptide synthesis using solid-phase techniques (Merrifield J.(1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesis may beperformed using manual techniques or by automation. Automated synthesismay be achieved, for example, using Applied Biosystems 431A PeptideSynthesizer (Perkin Elmer). Various fragments of BND may be chemicallysynthesized separately and combined using chemical methods to producethe full length molecule.

THERAPEUTICS

Based on the chemical and structural homology between BND-1 (SEQ IDNO:1) and human G25K (GI 183490), BND-2 (SEQ ID NO:3) and rab30 (GI1457955), and BND-3 and mouse GTP-binding protein (GI 240986), BNDappears to play a role in cell proliferation and signal transduction,and thus, may be used therapeutically for diseases and disordersinvolving these processes.

BND-1 appears to be involved in the regulation of the cell cycle, cellgrowth, cell signaling, and cellular cytoskeletal organization which arekey factors in neoplastic disease, arthritic disease, autoimmunedisease, inflammatory disease, and diseases of abnormal tissueproliferation. Expression of BND-1 is associated with aberrant tissueproliferation, autoimmune and inflammatory conditions. Therefore, in oneembodiment, vectors capable of expressing antisense to the nucleic acidsequence encoding BND-1 or a fragment or derivative thereof may beadministered to a subject to prevent or treat neoplastic disease,arthritic disease, autoimmune disease, inflammatory disease, anddiseases of abnormal tissues proliferation including, but not limitedto, cancer, leukemia, lymphoma, endometriosis, atherosclerosis, lupuserythematosus, and arthritis.

In another embodiment, antagonists or inhibitors of BND-1 may beadministered to a subject to treat any of the conditions describedabove. In one aspect, antibodies which are specific for BND-1 may beused directly as an antagonist, or indirectly as a targeting or deliverymechanism for administering a pharmaceutical agent to cells or tissuewhich express BND-1.

Based on the chemical and structural homology between BND-2 (SEQ IDNO:3) and rab30 (GI 1457955), and BND-3 and mouse GTP-binding protein(GI 240986), BND-2 and BND-3 appear to be involved in the regulation ofthe cellular vesicle targeting; membrane transfer and fusion; or proteinprocessing, targeting and secretion. Disorders and diseases involvingthese functions may include, but are not limited to, diminishedneurotransmitter and hormone secretion, lysosomal storage diseases,immunological disorders, and cancer.

In one embodiment, vectors expressing antisense to the nucleic acidsequence encoding BND-2 or a fragment or derivative thereof may beadministered to a subject to treat diseases or conditions associatedwith abnormal membrane transfer and fusion or abnormal proteinprocessing, targeting, and secretion.

In another embodiment, antagonists or inhibitors of BND-2 may beadministered to a subject to treat diminished neurotransmitter andhormone secretion, lysosomal storage diseases, immunological disorders,and cancer. In one aspect, antibodies which are specific for BND-2 maybe used directly as an antagonist, or indirectly as a targeting ordelivery mechanism for administering a pharmaceutical agent to cells ortissue which express BND-2.

In another embodiment, vectors expressing BND-2 may be administered to asubject to treat disorders resulting from a deficiency of BND-2expression which may include, but are not limited to, diminishedneurotransmitter and hormone secretion, lysosomal storage diseases,immunological disorders, and cancer.

In one embodiment, vectors expressing antisense to the nucleic acidsequence encoding BND-3 or a fragment or derivative thereof may beadministered to a subject to treat diseases or conditions associatedwith abnormal membrane transfer and fusion, abnormal protein processing,protein targeting and secretion.

In another embodiment, antagonists or inhibitors of BND-3 may beadministered to a subject to treat diminished neurotransmitter andhormone secretion, lysosomal storage diseases, immunological disorders,and cancer. In one aspect, antibodies which are specific for BND-3 maybe used directly as an antagonist, or indirectly as a targeting ordelivery mechanism for administering a pharmaceutical agent to cells ortissue which express BND-3.

In another embodiment, vectors expressing BND-3 may be administered to asubject to treat disorders resulting from a deficiency of BND-3expression which may include, but are not limited to, diminishedneurotransmitter and hormone secretion, lysosomal storage diseases,immunological disorders, and cancer.

In other embodiments, any of the therapeutic proteins, antagonists,antibodies, agonists, antisense sequences or vectors described above maybe administered in combination with other appropriate therapeuticagents. Selection of the appropriate agents for use in combinationtherapy may be made by one of ordinary skill in the art, according toconventional pharmaceutical principles. The combination of therapeuticagents may act synergistically to effect the treatment or prevention ofthe various disorders described above. Using this approach, one may beable to achieve therapeutic efficacy with lower dosages of each agent,thus reducing the potential for adverse side effects.

Agonists or inhibitors of BND-1, BND-2, or BND-3 may be produced usingmethods which are generally known in the art. In particular, purifiedBND may be used to produce antibodies or to screen libraries ofpharmaceutical agents to identify those which specifically bind BND.

Antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith BND or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, andrdinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

It is preferred that the peptides, fragments, or oligopeptides used toinduce antibodies to BND have an amino acid sequence consisting of atleast five amino acids, and more preferably at least 10 amino acids. Itis also preferable that they are identical to a portion of the aminoacid sequence of the natural protein, and they may contain the entireamino acid sequence of a small, naturally occurring molecule. Shortstretches of BND amino acids may be fused with those of another proteinsuch as keyhole limpet hemocyanin and antibody produced against thechimeric molecule.

Monoclonal antibodies to BND may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. etal. (1985) J Immunol. Methods 81:31-42; Cote, R. J. et al. (1983) Proc.Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al. (1985) Mol Cell Biol.62:109-120.

In addition, techniques developed for the production of "chimericantibodies", the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-55; Neuberger, M. S. et al. (1984) Nature312:604-8; Takeda, S. et al. (1985) Nature 314:452-4). Alternatively,techniques described for the production of single chain antibodies maybe adapted, using methods known in the art, to produce BND-specificsingle chain antibodies. Antibodies with related specificity, but ofdistinct idiotypic composition, may be generated by chain shuffling fromrandom combinatorial immunoglobin libraries (Burton D. R. (1991) Proc.Natl. Acad. Sci. 88:11120-3).

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-37; Winter, G. et al. (1991) Nature 349:293-9).

Antibody fragments which contain specific binding sites for BND may alsobe generated. For example, such fragments include, but are not limitedto, the F(ab')2 fragments which can be produced by pepsin digestion ofthe antibody molecule and the Fab fragments which can be generated byreducing the disulfide bridges of the F(ab')2 fragments. Alternatively,Fab expression libraries may be constructed to allow rapid and easyidentification of monoclonal Fab fragments with the desired specificity(Huse, W. D. et al. (1989) Science 254:1275-81).

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between BND and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering BND epitopes is preferred, but a competitive bindingassay may also be employed (Maddox, supra).

In another embodiment of the invention, the polynucleotides encodingBND, or any fragment thereof, or antisense molecules, may be used fortherapeutic purposes. In one aspect, antisense to the polynucleotideencoding BND may be used in situations in which it would be desirable toblock the transcription of mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding BND. Thus,antisense sequences may be used to modulate BND activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligomers or larger fragments, can bedesigned from various locations along the coding or control regions ofsequences encoding BND.

Expression vectors derived from retroviruses, adenovirus, herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensepolynucleotides of the gene encoding BND. These techniques are describedboth in Sambrook et al. (supra) and in Ausubel et al. (supra).

Genes encoding native BND can be turned off by transforming a cell ortissue with expression vectors which express high levels of thepolynucleotide, or fragment thereof, which encodes BND. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the genomic DNA,such vectors may continue to transcribe RNA molecules until they aredisabled by endogenous nucleases. Transient expression may last for amonth or more with a non-replicating vector and even longer ifappropriate replication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning antisense molecules, DNA, RNA, or PNA, to the control regionsof the gene encoding BND, i.e., the promoters, enhancers, and introns.Oligonucleotides derived from the transcription initiation site, e.g.,between positions -10 and +10 from the start site, are preferred.Similarly, inhibition can be achieved using "triple helix" base-pairingmethodology. Triple helix pairing is useful because it causes inhibitionof the ability of the double helix to open sufficiently for the bindingof polymerases, transcription factors, or regulatory molecules. Recenttherapeutic advances using triplex DNA have been described in theliterature (Gee, J. E. et al. (1994) In: Huber, B. E. and B. I. Carr,Molecular and Immunologic Approaches, Futura Publishing Co., Mt. Kisco,N.Y.). The antisense molecules may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding BND.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Antisense molecules and ribozymes of the invention may be prepared byany method known in the art for the synthesis of nucleic acid molecules.These include techniques for chemically synthesizing oligonucleotidessuch as solid phase phosphoramidite chemical synthesis. Alternatively,RNA molecules may be generated by in vitro and in vivo transcription ofDNA sequences encoding BND. Such DNA sequences may be incorporated intoa wide variety of vectors with suitable RNA polymerase promoters such asT7 or SP6. Alternatively, these cDNA constructs that synthesizeantisense RNA constitutively or inducibly can be introduced into celllines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the moleculeor the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection and by liposome injections may beachieved using methods which are well known in the art.

Any of the therapeutic methods described above may be applied to anysuitable subject including, for example, mammals such as dogs, cats,cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical composition, in conjunction with a pharmaceuticallyacceptable carrier, for any of the therapeutic effects discussed above.Such pharmaceutical compositions may consist of BND, antibodies to BND,mimetics, agonists, antagonists, or inhibitors of BND. The compositionsmay be administered alone or in combination with at least one otheragent, such as stabilizing compound, which may be administered in anysterile, biocompatible pharmaceutical carrier, including, but notlimited to, saline, buffered saline, dextrose, and water. Thecompositions may be administered to a patient alone, or in combinationwith other agents, drugs or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with a filler or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils such as sesame oil, or synthetic fatty acid esters, such asethyl oleate or triglycerides, or liposomes. Optionally, the suspensionmay also contain suitable stabilizers or agents which increase thesolubility of the compounds to allow for the preparation of highlyconcentrated solutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of BND, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually mice, rabbits, dogs, or pigs. The animal modelmay also be used to determine the appropriate concentration range androute of administration. Such information can then be used to determineuseful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example BND or fragments thereof, antibodies of BND,agonists, antagonists or inhibitors of BND, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio between therapeutic and toxic effects is thetherapeutic index, and it can be expressed as the ratio, LD50/ED50.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to atotal dose of about 1 g, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be pecific to particular cells,conditions, locations, etc.

DIAGNOSTICS

In another embodiment, antibodies which specifically bind BND may beused for the diagnosis of conditions or diseases characterized byexpression of BND, or in assays to monitor patients being treated withBND, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for BND includemethods which utilize the antibody and a label to detect BND in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

A variety of protocols including ELISA, RIA, and FACS for measuring BNDare known in the art and provide a basis for diagnosing altered orabnormal levels of BND expression. Normal or standard values for BNDexpression are established by combining body fluids or cell extractstaken from normal manmmalian subjects, preferably human, with antibodyto BND under conditions suitable for complex formation. The amount ofstandard complex formation may be quantified by various methods, butpreferably by photometric means. Quantities of BND expressed in subjectsamples, control and disease, from biopsied tissues are compared withthe standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encoding BNDmay be used for diagnostic purposes. The polynucleotides which may beused include oligonucleotide sequences, antisense RNA and DNA molecules,and PNAs. The polynucleotides may be used to detect and quantitate geneexpression in biopsied tissues in which expression of BND may becorrelated with disease. The diagnostic assay may be used to distinguishbetween absence, presence, and excess expression of BND, and to monitorregulation of BND levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding BND or closely related molecules, may be used to identifynucleic acid sequences which encode BND. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5' regulatory region, or a less specific region,e.g., especially in the 3' coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding BND, alleles, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably contain at least 50% of the nucleotides from any ofthe sequences encoding BND. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequences ofSEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 or from genomic sequenceincluding promoter, enhancer elements, and introns of the naturallyoccurring BND.

Means for producing specific hybridization probes for DNAs encoding BNDinclude the cloning of nucleic acid sequences encoding BND or BNDderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as 32P or 35S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

Polynucleotide sequences encoding BND may be used for the diagnosis ofconditions or diseases which are associated with expression of BND.Examples of such conditions or diseases include, but are not limited to,tumors, leukemia, lymphoma, hemolytic anemia, lupus erythematosus,rheumatoid arthritis, endometriosis, diminished neurotransmitter andhormone secretion, lysosomal storage diseases, immunological disorders,and cancer. The polynucleotide sequences encoding BND may be used inSouthern or northern analysis, dot blot, or other membrane-basedtechnologies; in PCR technologies; or in dip stick, pin, ELISA or chipassays utilizing fluids or tissues from patient biopsies to detectaltered BND expression. Such qualitative or quantitative methods arewell known in the art.

In a particular aspect, the nucleotide sequences encoding BND may beuseful in assays that detect activation or induction of various cancers,particularly those mentioned above. The nucleotide sequences encodingBND may be labeled by standard methods, and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the biopsied or extracted sample issignificantly altered from that of a comparable control sample, thenucleotide sequences have hybridized with nucleotide sequences in thesample, and the presence of altered levels of nucleotide sequencesencoding BND in the sample indicates the presence of the associateddisease. Such assays may also be used to evaluate the efficacy of aparticular therapeutic treatment regimen in animal studies, in clinicaltrials, or in monitoring the treatment of an individual patient.

In order to provide a basis for the diagnosis of disease associated withexpression of BND, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, which encodes BND, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withthose from an experiment where a known amount of a substantiallypurified polynucleotide is used. Standard values obtained from normalsamples may be compared with values obtained from samples from patientswho are symptomatic for disease. Deviation between standard and subjectvalues is used to establish the presence of disease.

Once disease is established and a treatment protocol is initiated,hybridization assays may be repeated on a regular basis to evaluatewhether the level of expression in the patient begins to approximatethat which is observed in the normal patient. The results obtained fromsuccessive assays may be used to show the efficacy of treatment over aperiod ranging from several days to months.

With respect to cancer, the presence of a relatively low amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding BND may involve the use of PCR. Such oligomers may bechemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5'->3') and another withantisense (3'<-5'), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of BNDinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and standard curves onto which the experimentalresults are interpolated (Melby, P. C. et al. (1993) J. Immunol.Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 229-236).The speed of quantitation of multiple samples may be accelerated byrunning the assay in an ELISA format where the oligomer of interest ispresented in various dilutions and a spectrophotometric or calorimetricresponse gives rapid quantitation.

In another embodiment of the invention, the nucleic acid sequence whichencodes BND may also be used to generate hybridization probes which areuseful for mapping the naturally occurring genomic sequence. Thesequence may be mapped to a particular chromosome or to a specificregion of the chromosome using well known techniques. Such techniquesinclude FISH, FACS, or artificial chromosome constructions, such asyeast artificial chromosomes, bacterial artificial chromosomes,bacterial P1 constructions or single chromosome CDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

FISH (as described in Verma, R. S. et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding BND on a physical chromosomal map and a specific disease ,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques such as linkage analysis using established chromosomalmarkers may be used for extending genetic maps. Often the placement of agene on the chromosome of another mammalian species, such as mouse, mayreveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms, or parts thereof, by physical mapping. This providesvaluable information to investigators searching for disease genes usingpositional cloning or other gene discovery techniques. Once the diseaseor syndrome has been crudely localized by genetic linkage to aparticular genomic region, for example, AT to 11q22-23 (Gatti, R. A. etal. (1988) Nature 336:577-580), any sequences mapping to that area mayrepresent associated or regulatory genes for further investigation. Thenucleotide sequence of the subject invention may also be used to detectdifferences in the chromosomal location due to translocation, inversion,etc. among normal, carrier, or affected individuals.

In another embodiment of the invention, BND, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, between BNDand the agent being tested, may be measured.

Another technique for drug screening which may be used provides for highthroughput screening of compounds having suitable binding affinity tothe protein of interest as described in published PCT applicationWO84/03564. In this method, as applied to BND large numbers of differentsmall test compounds are synthesized on a solid substrate, such asplastic pins or some other surface. The test compounds are reacted withBND, or fragments thereof, and washed. Bound BND is then detected bymethods well known in the art. Purified BND can also be coated directlyonto plates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding BND specificallycompete with a test compound for binding BND. In this manner, theantibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with BND.

In additional embodiments, the nucleotide sequences which encode BND maybe used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES I DNA Library Construction

TESTNOT01

Tissue was obtained from a 37-year-old Caucasian male (lot no. 94-267);Keystone Skin Bank, International Institute for the Advancement ofMedicine (Exton, Pa.). The tissue was flash frozen, ground using amortar and pestle and was lysed immediately in buffer containingguanidinium isothiocyanate. Lysis was followed by several phenolchloroform extractions and ethanol precipitation. Polyadenylated RNA wasisolated using biotinylated oligo d(T) primer and streptavidin coupledto a paramagnetic particle (Promega Corp., Madison, Wis.) and sent toStratagene.

Stratagene prepared the cDNA library using oligo d(T) priming. Syntheticadapter oligonucleotides were ligated onto the cDNA molecules enablingthem be inserted into the UNI-ZAP™ vector system (Stratagene).

The quality of the cDNA library was screened using DNA probes, and thePBLUESCRIPT® phagemid (Stratagene) was excised. Subsequently, thecustom-constructed library phage particles were infected into E. colihost strain XLI-BLUE® (Stratagene). Alternative unidirectional vectorsmight include, but are not limited to, pcDNAI (Invitrogen, San Diego,Calif.) and pSHlox-1 (Novagen, Madison, Wis.).

PROSNOT01

The prostate tissue used for library construction was obtained from a 50year-old male. The tissue was flash frozen, ground using a mortar andpestle, extracted four times with acid phenol pH 4.0, and centrifugedover a CsCl cushion using an Beckman SW28 rotor in a Beckman L8-70MUltracentrifuge (Beckman Instruments). The RNA was precipitated using0.3M sodium acetate and 2.5 volumes of ethanol, resuspended in water,and DNase treated for 15 min at 37° C. The RNA was isolated using theQiagen Oligotex kit (QIAGEN Inc, Chatsworth Calif.) and used toconstruct the CDNA library.

PGANNOT01

The tissue used for paraganglion cDNA library construction was obtainedfrom a 46 year-old male. The frozen tissue was homogenized and lysed inguanidinium isothiocyanate solution using a Brinkmann HomogenizerPolytron PT-3000 (Brinkmann Instruments, Westbury N.J.). The lysate wascentrifuged over a 5.7M CsCl cushion using an Beckman SW28 rotor in aBeckman L8-70M Ultracentrifuge (Beckman Instruments) for 18 hours at25,000 rpm at ambient temperature. The RNA was extracted twice with acidphenol pH 4.0 following Stratagene's RNA isolation protocol,precipitated using 0.3M sodium acetate and 2.5 volumes of ethanol,resuspended in DEPC-treated water, and DNase treated for 15 min at 37°C. The reaction was stopped with an equal volume of acid phenol and theRNA was isolated using the Qiagen Oligotex kit and used to construct thecDNA library.

The RNA was handled according to the recommended protocols in theSuperScript Plasmid System for cDNA Synthesis and Plasmid Cloning(catalog #18248-013; Gibco/BRL), and cDNAs were ligated into PSPORTI.The plasmid PSPORTI was subsequently transformed into DH5A™ competentcells (Cat. #18258-012, Gibco/BRL).

II Isolation of cDNA Clones

TESTNOT01

The phagemid forms of the individual cDNA clones were obtained by the invivo excision process, in which the host bacterial strain wasco-infected with both the library phage and an f1 helper phage.Polypeptides or enzymes derived from both the library-containing phageand the helper phage nicked the DNA, initiated new DNA synthesis fromdefined sequences on the target DNA, and created a smaller, singlestranded circular phagemid DNA molecule that included all DNA sequencesof the pBluescript phagemid and the cDNA insert. The phagemid DNA wasreleased from the cells and purified, and used to reinfect fresh hostcells (SOLR, Stratagene) where double stranded DNA was produced. Becausethe phagemid carries the gene for β-lactamase, the newly transformedbacteria were selected on medium containing ampicillin.

Phagemid DNA was purified using the QIAWELL™ -8 Plasmid Purificationsystem (QIAGEN). The DNA was eluted from the purification resin andprepared for DNA sequencing and other analytical manipulations.

PROSNOT01 and PGANNOT01

Plasmid DNA was released from the cells and purified using the MiniprepKit (Catalogue #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Catalog #22711, LIFE TECHNOLOGIES™,Gaithersburg Md.) with carbenicillin at 25 mg/L and glycerol at 0.4%; 2)the bacteria were cultured for 24 hours after the wells were inoculatedand then lysed with 60 μl of lysis buffer; 3) a centrifugation stepemploying the Beckman GS-6R @2900 rpm for 5 min was performed before thecontents of the block were added to the primary filter plate; and 4) theoptional step of adding isopropanol to TRIS buffer was not routinelyperformed. After the last step in the protocol, samples were transferredto a Beckman 96-well block for storage.

The cDNAs for all three libraries were sequenced by the method of SangerF and A. R. Coulson (1975; J Mol Biol 94:441f), using a Hamilton MicroLab 2200 (Hamilton, Reno, Nev.) in combination with four Peltier ThermalCyclers (PTC200 from MJ Research, Watertown Mass.) and AppliedBiosystems 377 or 373 DNA Sequencing Systems (Perkin Elmer), and readingframe was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT™ 670sequence analysis system. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles, Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT-670 sequence analysis system using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul, S. F. et al. (1990) J.Mol. Biol. 215:403-410), was used to search for local sequencealignments. BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs. BLAST is useful for matches which donot contain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound (Sambrook et al., supra).

Analogous computer techniques using BLAST (Altschul, S. F. 1993 and1990, supra) are used to search for identical or related molecules innucleotide databases such as GenBank or the LIFESEQ™ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

The basis of the search is the product score which is defined as:

    % sequence identity×% maximum BLAST score/100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1-2% error;and at 70, the match will be exact. Homologous molecules are usuallyidentified by selecting those which show product scores between 15 and40, although lower scores may identify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding BND occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V Extension of Polynucleotides Encoding BND to Full Length or to RecoverRegulatory Sequences

Polynucleotides encoding BND (SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6)are used to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5' or 3', intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence "outward" generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

The original, selected cDNA libraries, or a human genomic library areused to extend the sequence; the latter is most useful to obtain 5'upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

By following the instructions for the XL-PCR kit (Perkin Elmer) andthoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters:

Step 1 94° C. for 1 min (initial denaturation)

Step 2 65° C. for 1 min

Step 3 68° C. for6 min

Step 4 94° C. for 15 sec

Step 5 65° C. for 1 min

Step 6 68° C. for 7 min

Step 7 Repeat step 4-6 for 15 additional cycles

Step 8 94° C. for 15 sec

Step 9 65° C. for 1 min

Step 10 68° C. for 7:15 min

Step 11 Repeat step 8-10 for 12 cycles

Step 12 72° C. for 8 min

Step 13 4° C. (and holding)

A 5-10 μl aliquot of the reaction mixture is analyzed by electrophoresison a low concentration (about 0.6-0.8%) agarose mini-gel to determinewhich reactions were successful in extending the sequence. Bands thoughtto contain the largest products are selected and removed from the gel.Further purification involves using a commercial gel extraction methodsuch as QIAQuick Kit (Qiagen Inc.). After recovery of the DNA, Kienowenzyme is used to trim single-stranded, nucleotide overhangs creatingblunt ends which facilitate religation and cloning.

After ethanol precipitation, the products are redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150 μl of liquid LB/2× Carbmedium placed in an individual well of an appropriate,commercially-available, sterile 96-well microtiter plate. The followingday, 5 μl of each overnight culture is transferred into a non-sterile96-well plate and after dilution 1:10 with water, 5 μl of each sample istransferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction areadded to each well. Amplification is performed using the followingconditions:

Step 1 94° C. for 60 sec

Step 2 94° C. for 20 sec

Step 3 55° C. for 30 sec

Step 4 72° C. for 90 sec

Step 5 Repeat steps 2-4 for an additional 29 cycles

Step 6 72° C. for 180 sec

Step 7 4° C. (and holding)

Aliquots of the PCR reactions are run on agarose gels together withmolecular weight markers. The sizes of the PCR products are compared tothe original partial cDNAs, and appropriate clones are selected, ligatedinto plasmid, and sequenced.

VI Labeling and Use of Hybridization Probes

Hybridization probes derived from SEQ ID NO:2, SEQ ID NO:4, or SEQ IDNO:6 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although thelabeling of oligonucleotides, consisting of about 20 base-pairs, isspecifically described, essentially the same procedure is used withlarger cDNA fragments. Oligonucleotides are designed usingstate-of-the-art software such as OLIGO 4.06 (National Biosciences),labeled by combining 50 pmol of each oligomer and 250 μCi of γ-³² -P!adenosine triphosphate (Amersham) and T4 polynucleotide kinase (DuPontNEN®, Boston, Mass.). The labeled oligonucleotides are substantiallypurified with Sephadex G-25 superfine resin column (Pharmacia & Upjohn).A portion containing 10⁷ counts per minute of each of the sense andantisense oligonucleotides is used in a typical membrane basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II;DuPont NEN®).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1× salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots in a Phosphoimagercassette (Molecular Dynamics, Sunnyvale, Calif.) for several hours,hybridization patterns are compared visually.

VII Antisense Molecules

Antisense molecules to the sequence encoding BND, or any part thereof,is used to inhibit in vivo or in vitro expression of naturally occurringBND. Although use of antisense oligonucleotides, comprising about 20base-pairs, is specifically described, essentially the same procedure isused with larger cDNA fragments. An oligonucleotide based on thesequences encoding BND is used to inhibit expression of naturallyoccurring BND. The complementary oligonucleotide is designed from themost unique 5' sequence as shown in and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of a transcript encoding BND bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5' sequence of SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6,an effective antisense oligonucleotide includes any 15-20 nucleotidesspanning the region which translates into the signal or 5' codingsequence of the polypeptide as shown in FIGS. 1A and 1B, 2A and 2B, and3A and 3B.

VIII Expression of BND

Expression of BND is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, PSPORTI previously used for thegeneration of the cDNA library is used to express BND in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

Induction of an isolated, transformed bacterial strain with IPTG usingstandard methods produces a fusion protein which consists of the firsteight residues of β-galactosidase, about 5 to 15 residues of linker, andthe full length protein. The signal residues direct the secretion of BNDinto the bacterial growth media which can be used directly in thefollowing assay for activity.

IX Demonstration of BND Activity

GTP-binding activity is assayed by incubating varying amounts of BNDprotein for 10 minutes at 30° C. in 50 mM Tris buffer, pH 7.5,containing 1 mM dithiothreitol, 1 mM EDTA, 1 μM (a-³² P), in the absenceor presence of 100 μM of the following compounds: GTP, GDP, GTPγS, ATP,CTP, UTP, and TTP. Samples are passed through nitrocellulose filters andwashed twice with a buffer consisting of 50 mM Tris-HCL, pH 7.8, 1 mMNaN₃, 10 mM MgCl₂, 1 mM EDTA, 0.5 mM dithiothreitol, 0.01 mM PMSF, and200 mM NaCl. The filter-bound counts are determined by liquidscintillation. To determine GTPase activity, BND protein is incubatedfor 10 minutes at 37° C. in 50 mM Tris-HCL buffer, pH 7.8, containing 1mM dithiothreitol, 2 mM EDTA, 10 μM (a-³² P), and 1 μM H-rab protein.GTPase activity is initiated by adding MgCl₂ to a final concentration of10 mM. Samples are removed at various time points, mixed with an equalvolume of ice-cold 0.5 mM EDTA, and frozen. Aliquots are spotted ontopolyethyleneimine-cellulose thin layer chromatography plates, which aredeveloped in 1M LiCl, dried, and autoradiographed.

X Production of BND Specific Antibodies

BND that is substantially purified using PAGE electrophoresis (Sambrook,supra), or other purification techniques, is used to immunize rabbitsand to produce antibodies using standard protocols. The amino acidsequence deduced from SEQ ID NO:2, SEQ ID NO:4, or SEQ ID NO:6 isanalyzed using DNASTAR software (DNASTAR Inc) to determine regions ofhigh immunogenicity and a corresponding oligopolypeptide is synthesizedand used to raise antibodies by means known to those of skill in theart. Selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions, is described by Ausubel et al.(supra), and others.

Typically, the oligopeptides are 15 residues in length, synthesizedusing an Applied Biosystems Peptide Synthesizer Model 431A usingfmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH, Sigma,St. Louis, Mo.) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimideester (MBS; Ausubel et al., supra). Rabbits are immunized with theoligopeptide-KLH complex in complete Freund's adjuvant. The resultingantisera are tested for antipeptide activity, for example, by bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radioiodinated, goat anti-rabbitIgG.

XI Purification of Naturally Occurring BND Using Specific Antibodies

Naturally occurring or recombinant BND is substantially purified byimmunoaffinity chromatography using antibodies specific for BND. Animmunoaffinity column is constructed by covalently coupling BND antibodyto an activated chromatographic resin, such as CnBr-activated Sepharose(Pharmacia & Upjohn). After the coupling, the resin is blocked andwashed according to the manufacturer's instructions.

Media containing BND is passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof BND (e.g., high ionic strength buffers in the presence of detergent).The column is eluted under conditions that disrupt antibody/BND binding(eg, a buffer of pH 2-3 or a high concentration of a chaotrope, such asurea or thiocyanate ion), and BND is collected.

XII Identification of Molecules Which Interact with BND

BND or biologically active fragments thereof are labeled with ¹²⁵ IBolton-Hunter reagent (Bolton, A. E. and W. M. Hunter (1973) Biochem. J.133: 529-39). Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled BND, washed and anywells with labeled BND complex are assayed. Data obtained usingdifferent concentrations of BND are used to calculate values for thenumber, affinity, and association of BND with the candidate molecules.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the described modes forcarrying out the invention which are obvious to those skilled inmolecular biology or related fields are intended to be within the scopeof the following claims.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 9                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 181 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: SEQ ID NO:1                                                      (B) CLONE: 113700                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       MetThrAsnIleGlyValSerTyrThrProAsnGlyTyrProThrGlu                              151015                                                                        TyrIleProThrAlaPheAspAsnPheSerAlaValValSerValAsp                              202530                                                                        GlyArgProValArgLeuGlnLeuCysAspThrAlaGlyGlnAspGlu                              354045                                                                        PheAspLysLeuArgProLeuCysTyrThrAsnThrAspIlePheLeu                              505560                                                                        LeuCysPheSerValValSerProSerSerPheGlnAsnValSerGlu                              65707580                                                                      LysTrpValProGluIleArgCysHisCysProLysAlaProIleIle                              859095                                                                        LeuValGlyThrGlnSerAspLeuArgGluAspValLysValLeuIle                              100105110                                                                     GluLeuAspLysCysLysGluLysProValProGluGluAlaAlaLys                              115120125                                                                     LeuCysAlaGluGluIleLysAlaAlaSerTyrIleGluCysSerAla                              130135140                                                                     LeuThrGlnLysAsnLeuLysGluValPheAspAlaAlaIleValAla                              145150155160                                                                  GlyIleGlnTyrSerAspThrGlnGlnGlnProLysLysSerLysSer                              165170175                                                                     ArgThrProAspLys                                                               180                                                                           (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 719 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: SEQ ID NO:2                                                      (B) CLONE: 113700                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       CGGGCGTCAAGTGGGTCCCCGGTCGGAAAAAAGCGCGGTTTGGATGACAAACATTGGGGT60                GAGCTACACCCCCAACGGCTACCCCACCGAGTACATCCCTACTGCCTTCGACAACTTCTC120               CGCGGTGGTGTCTGTGGATGGGCGGCCCGTGAGACTCCAACTCTGTGACACTGCCGGACA180               GGATGAATTTGACAAGCTGAGGCCTCTCTGCTACACCAACACAGACATCTTCCTGCTCTG240               CTTCAGTGTCGTGAGCCCCTCATCCTTCCAGAACGTCAGTGAGAAATGGGTGCCGGAGAT300               TCGATGCCACTGTCCCAAAGCCCCCATCATCCTAGTTGGAACGCAGTCGGATCTCAGAGA360               AGATGTCAAAGTCCTCATTGAGTTGGACAAATGCAAAGAAAAGCCAGTGCCTGAAGAGGC420               GGCTAAGCTGTGCGCCGAGGAAATCAAAGCCGCCTCCTACATCGAGTGTTCAGCCTTGAC480               TCAAAAAAACCTCAAAGAGGTCTTTGATGCAGCCATCGTCGCTGGCATTCAATACTCGGA540               CACTCAGCAACAGCCAAAGAAGTCTAAAAGCAGGACTCCAGATAAATGAAAAACCTCTCC600               AAGTCCTGGTGGAAGAAGTACTGCTGTTTCGTATGATGCTGGCAAGACACCCAGAAAGGC660               TATTTTCAGATGAATCGATATTAGAGCTTATTAGTGAAACAACTCTTTTACTGGTGGAC719                (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 186 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: SEQ ID NO: 3                                                     (B) CLONE: 538177                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       MetValLysLeuAlaAlaLysCysIleLeuAlaGlyAspProAlaVal                              151015                                                                        GlyLysThrAlaLeuAlaGlnIlePheArgSerAspGlyAlaHisPhe                              202530                                                                        GlnLysSerTyrThrLeuThrThrGlyMetAspLeuValValLysThr                              354045                                                                        ValProValProAspThrGlyAspSerValGluLeuPheIlePheAsp                              505560                                                                        SerAlaGlyLysGluLeuPheSerGluMetLeuAspLysLeuTrpGlu                              65707580                                                                      SerProAsnValLeuCysLeuValTyrAspValThrAsnGluGluSer                              859095                                                                        PheAsnAsnCysSerLysTrpLeuGluLysAlaArgSerGlnAlaPro                              100105110                                                                     GlyIleSerLeuProGlyValLeuValGlyAsnLysThrAspLeuAla                              115120125                                                                     GlyArgArgAlaValAspSerAlaGluAlaArgAlaTrpAlaLeuGly                              130135140                                                                     GlnGlyLeuGluCysPheGluThrSerValLysGluMetGluAsnPhe                              145150155160                                                                  GluAlaProPheHisCysLeuAlaLysGlnPheHisGlnLeuTyrArg                              165170175                                                                     GluLysValGluValPheArgAlaLeuAla                                                180185                                                                        (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 779 base pairs                                                    (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: SEQ ID NO:4                                                      (B) CLONE: 538177                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       CCGGANTCGTCCTTCCTTGAACCCTCTGGGCCAGCCCAGGGCCCGAAGCCCACCCACTCG60                CNTCTCTAGCAGCCGCTCTTGTCCTCTGGGTACGGCTCGCGGGAGTTTTGGTTACCATGG120               TGAAGCTGGCAGCCAAATGCATCCTGGCAGGAGACCCAGCAGTGGGCAAGACCGCCCTNG180               CACAGATCTTCCGCAGTGATGGAGCCCATTTCCAGAAAAGCTACACCCTGACAACAGGAA240               TGGATTTGGTGGTGAAGACAGTGCCAGTTCCTGACACGGGAGACAGTGTGGAACTCTTCA300               TTTTTGACTCTGCTGGCAAGGAGCTGTTTTCGGAAATGCTGGATAAATTGTGGGAGAGTC360               CCAATGTCTTATGTCTCGTCTATGATGTGACCAATGAAGAATCCTTCAACAACTGCAGCA420               AGTGGCTGGAGAAGGCTCGGTCACAGGCTCCAGGCATCTCTCTCCCAGGTGTTTTAGTTG480               GGAACAAGACAGACCTGGCCGGCAGACGAGCAGTGGACTCAGCTGAGGCCCGGGCATGGG540               CGCTGGGCCAGGGCCTGGAATGTTTTGAAACATCCGTGAAAGAGATGGAAAACTTCGAAG600               CCCCTTTCCACTGCCTTGCCAAGCAGTTCCACCAGCTGTACCGGGAGAAGGTGGAGGTTT660               TCCGGGCCCTGGCATGACGAGCTGGAGCAGATCGTGCTGCACAACCGGAGAAGACAGAAT720               TACCTCTGCTCTTTTAATATATAATGATGGCTTTAAATAAAATTAGGAGAAAATGTAAA779                (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 169 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: SEQ ID NO:5                                                      (B) CLONE: 627051                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       MetGluArgPheGluValLeuGlyIleProPheSerLeuGlnLeuTrp                              151015                                                                        AspThrAlaGlyGlnGluArgPheLysCysIleAlaSerThrTyrTyr                              202530                                                                        ArgGlyAlaGlnAlaIleIleIleValPheAsnLeuAsnAspValAla                              354045                                                                        SerLeuGluHisThrLysGlnTrpLeuAlaAspAlaLeuLysGluAsn                              505560                                                                        AspProSerSerValLeuLeuPheLeuValGlySerLysLysAspLeu                              65707580                                                                      SerThrProAlaGlnTyrAlaLeuMetGluLysXaaAlaLeuGlnVal                              859095                                                                        AlaGlnGlyMetLysAlaGluTyrTrpAlaValSerSerLeuThrGly                              100105110                                                                     GluAsnValArgGluPhePhePheArgValAlaAlaLeuThrPheGlu                              115120125                                                                     AlaAsnValLeuAlaGluLeuGluLysSerGlyAlaArgArgIleGly                              130135140                                                                     AspValValArgIleAsnSerAspAspSerAsnLeuTyrLeuThrAla                              145150155160                                                                  SerLysLysLysProThrCysCysPro                                                   165                                                                           (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1255 base pairs                                                   (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: SEQ ID NO:6                                                      (B) CLONE: 627051                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       CAGCAGTCTNCGATTCCCCATNACCAATTCGGCTNGGGTCTNCGCGGGCCCGGCCCCCAC60                CAGACGGGACTNCCCGNCCCCAATTNGCGGCCGAAGAGTCTCCTCGCCCCAGAGTCATCT120               TNGGGACGACCAGGGCCCGGGTGATTTTGGGCTCGCCGCGGCCCCKGGTGATTGTTTCAT180               CTCCGTGGCCCGCGGTGGTCGTAGCGTCTCCGAGACCGCGGACTCCCGTAGGTCCCCGTG240               GCCCCGAGTTGTAGTCGGGACACCCCGGCCGCGGGTGATCGTCGGGTCTCCAAGCGCCCG300               GGTCGCTGACGCGGATCCGGCCTYGGCGCCTTCTCAGGGGCGCCCTGCAAGGCCGCAGGC360               AGGATGAACATTCTGGCACCCGTGCGGAGGGATCGCGTCCTGGCGGACTGCCCCAGTGCC420               TGAGGAAGGAGGCCGCTTTGCACGGGCACAAAGACTTCCACCCCCGCGTCACCTGCGCCT480               GCCAGGAGCACCGGACAGGCACCGTGGGATTTAAGATCTCCAAGGTCATTGTGGTGGGGG540               ACCTGTCGGTGGGGAAGACTTGCCTCATTAATAGGTTCTGCAAAGACACCTTTGATAAGA600               ATTACAAGGCCACCATTGGAGTGGACTTCGAGATGGAACGATTTGAGGTGCTGGGCATTC660               CCTTCAGTTTGCAGCTTTGGGATACCGCTGGGCAGGAGAGGTTCAAATGCATTGCATCAA720               CCTACTATAGAGGAGCTCAAGCCATCATCATTGTCTTCAACCTGAATGATGTGGCATCTC780               TGGAACATACCAAGCAGTGGCTGGCCGATGCCCTGAAGGAGAATGACCCTTCCAGTGTGC840               TTCTCTTCCTTGTAGGTTCCAAGAAGGATCTGAGTACCCCTGCTCAGTATGCGCTGATGG900               AGAAAGAMGCCCTCCAGGTGGCCCAGGGGATGAAGGCTGAGTACTGGGCAGTCTCATCTC960               TCACTGGTGAGAATGTCCGAGAATTCTTCTTCCGTGTGGCAGCACTGACCTTTGAGGCCA1020              ATGTGCTGGCTGAGCTGGAGAAATCGGGGGCTCGACGCATTGGGGATGTTGTCCGCATCA1080              ACAGTGATGACAGCAACCTSTACCTAACTGCCAGCAAGAAGAAGCCCACATGTTGCCCAT1140              GAGGGCTGAGGAGACTGTTCAGAGACTGCCCAGCCCTAGGGCACTGTGCCACCCTCATTY1200              CTTCAGAGNTTGACCCCTGGGNGANTTGCANTGACTTTATTCAGACCAAAGAGGT1255                   (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 191 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 183490                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       MetGlnThrIleLysCysValValValGlyAspGlyAlaValGlyLys                              151015                                                                        ThrCysLeuLeuIleSerTyrThrThrAsnLysPheProSerGluTyr                              202530                                                                        ValProThrValPheAspAsnTyrAlaValThrValMetIleGlyGly                              354045                                                                        GluProTyrThrLeuGlyLeuPheAspThrAlaGlyGlnGluAspTyr                              505560                                                                        AspArgLeuArgProLeuSerTyrProGlnThrAspValPheLeuVal                              65707580                                                                      CysPheSerValValSerProSerSerPheGluAsnValLysGluLys                              859095                                                                        TrpValProGluIleThrHisHisCysProLysThrProPheLeuLeu                              100105110                                                                     ValGlyThrGlnIleAspLeuArgAspAspProSerThrIleGluLys                              115120125                                                                     LeuAlaLysAsnLysGlnLysProIleThrProGluThrAlaGluLys                              130135140                                                                     LeuAlaArgAspLeuLysAlaValLysTyrValGluCysSerAlaLeu                              145150155160                                                                  ThrGlnLysGlyLeuLysAsnValPheAspGluAlaIleLeuAlaAla                              165170175                                                                     LeuGluProProGluProLysLysSerArgArgCysValLeuLeu                                 180185190                                                                     (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 203 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 1457955                                                            (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       MetSerMetGluAspTyrAspPheLeuPheLysIleValLeuIleGly                              151015                                                                        AsnAlaGlyValGlyLysThrCysLeuValArgArgPheThrGlnGly                              202530                                                                        LeuPheProProGlyGlnGlyAlaThrIleGlyValGlyPheMetIle                              354045                                                                        LysThrValGluIleAsnGlyGluLysValLysLeuGlnIleTrpAsp                              505560                                                                        ThrAlaGlyGlnGluArgPheArgSerIleThrGlnSerTyrTyrArg                              65707580                                                                      SerAlaAsnAlaLeuIleLeuThrTyrAspIleThrCysGluGluSer                              859095                                                                        PheArgCysLeuProGluTrpLeuArgGluIleGluGlnTyrAlaSer                              100105110                                                                     AsnLysValIleThrValLeuValGlyAsnLysIleAspLeuAlaGlu                              115120125                                                                     ArgArgGluValSerGlnGlnArgAlaGluGluPheSerGluAlaGln                              130135140                                                                     AspMetTyrTyrLeuGluThrSerAlaLysGluSerAspAsnValGlu                              145150155160                                                                  LysLeuPheLeuAspLeuAlaCysArgLeuIleSerGluAlaArgGln                              165170175                                                                     AsnThrLeuValAsnAsnValSerSerProLeuProGlyGluGlyLys                              180185190                                                                     SerIleSerTyrLeuThrCysCysAsnPheAsn                                             195200                                                                        (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 208 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (vii) IMMEDIATE SOURCE:                                                       (A) LIBRARY: GenBank                                                          (B) CLONE: 240986                                                             (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       AsnSerLysValIleValValGlyAspLeuSerValGlyLysThrCys                              151015                                                                        LeuIleAsnArgPheCysLysAspThrPheAspLysAsnTyrLysAla                              202530                                                                        ThrIleGlyValAspPheGluMetGluArgPheGluValLeuGlyVal                              354045                                                                        ProPheSerLeuGlnLeuTrpAspThrAlaGlyGlnGluArgPheLys                              505560                                                                        CysIleAlaSerThrTyrTyrArgGlyAlaGlnAlaIleIleIleVal                              65707580                                                                      PheAsnLeuAsnAspValAlaSerLeuGluHisThrLysGlnTrpLeu                              859095                                                                        ThrAspAlaLeuLysGluAsnAspProSerAsnValLeuLeuPheLeu                              100105110                                                                     ValGlySerLysLysAspLeuSerThrProAlaGlnTyrSerLeuMet                              115120125                                                                     GluLysAspAlaLeuLysValAlaGlnGluIleLysAlaGluTyrTrp                              130135140                                                                     AlaValSerSerLeuThrGlyGluAsnValArgGluPhePhePheArg                              145150155160                                                                  ValAlaAlaLeuThrPheGluAlaAsnValLeuAlaAspValGluLys                              165170175                                                                     SerGlyAlaArgHisIleAlaAspValValArgIleAsnSerAspAsp                              180185190                                                                     LysAsnLeuTyrLeuThrAlaSerLysLysLysAlaThrCysCysPro                              195200205                                                                     __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide sequenceencoding a polypeptide comprising the amino acid sequence of SEQ IDNO:1.
 2. A hybridization probe comprising the polynucleotide sequence ofclaim
 1. 3. An isolated and purified polynucleotide sequence comprisingSEQ ID NO:2.
 4. A polynucleotide sequence which is fully complementaryto SEQ ID NO:2.
 5. A hybridization probe comprising the polynucleotidesequence of claim
 4. 6. An expression vector containing thepolynucleotide sequence of claim
 1. 7. A host cell containing the vectorof claim 6.